METHOD OF IMMUNOSIGNATURE IN DIFFERENTIAL DIAGNOSIS OF AUTISM SPECTRUM DISORDERS. A PILOT STUDY

A large and diverse repertoire of antibodies encodes the history of past immunological experience, creating a global network of the body’s regulation system. In this article, we propose to use a peptide microarray (“immunosignature”) for evaluating global individual antibody patterns and bioinformatic data analysis for differential diagnosis of autism spectrum disorders. The peptide microarray consists of 124 000 antigen mimetics with random sequences covalently bound to the surface of the glass slide. A drop of plasma is tested for the presence of antibodies of distinct specificity, by measuring their binding to each antigen mimetic in the microarray detectable by fluorescent staining with secondary IgG antibodies, and this reaction is registered by laser activation assay. For bioinformatic analysis, we used the files of digitalized fluorescence intensity data, which presented the reactivity of plasma antibodies bound to antigen mimetics. Data processing was carried out by packages of the Bioconductor project for the R software environment to perform statistical evaluation. At the stage of primary data processing, the quantile normalization was used in order to equalize the distributions of antibodies’ reactivity. The sample data and other necessary information were combined into the discrete ExpessionSet container files. To compare the control and experimental groups, the Welch’s one-way ANOVA (for unequal variances) was used. The obtained estimates of the mean value differences and statistical significance of P levels were used further for constructing a volcano diagram, in order of ranking and selecting the most promising antibody reactivity parameters. For differential diagnosis of autism, and evaluation of diagnostic significance of the immunosignature method, a heatmap was constructed. The standardized values of the logarithms of antibody reactivity, and the results of the hierarchical cluster analysis performed by the Ward method using Pearson correlation, as a measure of similarity were used in constructing the heatmap. As a result of the bioinformatiс analysis of the data, 73 antibodies were selected whose reactivity had statistically significant differences in groups of children with autism and normally developing children. These antibodies were used for differential diagnosis, the value of which was determined in the heatmap construction. It was found that the group of children with autism spectrum disorders by the antibody reactivity exhibits marked heterogeneity, and consists of at least two subgroups. In addition, 60 antibodies in children with autism showed predominantly medium and low reactivity, i.e. these antibodies had a weak binding power with antigenic mimetics, and only 13 antibodies showed high reactivity. In general, diagnostic specificity of the autism spectrum disorders using immunosignature approach was 96.0% (95% CI 82.8 to 99.6%), sensitivity was 78.3% (95% CI 64.9 to 88.2%), and diagnostic efficiency was 82.7%. Our pilot study allows us to propose a method of immunosignature for differential diagnosis of autism and, possibly, to expand our understanding of autism spectrum disorders

Assessment of the size and shape of berries using the ImageJ program on the example of honeysuckle

Background. Digital technologies are increasingly used in agriculture to solve a variety of problems. However, in horticulture and industrial production of fruit and berry crops, qualitative evaluation and scoring of the most important morphological indicators of fruits are still common, and measurements are carried out manually. The aim of this study was to develop an algorithm for using the ImageJ package for quick and accurate measurements of the size and shape of berries.Materials and methods. The material included 190 berries of 3 blue honeysuckle (Lonicera caerulea L.) cultivars: ‘Amazonka’, ‘Lazurit’, and ‘Lenita’. The berries were laid out on a white sheet of paper with a ruler on top of the glass and photographed with additional lighting from below. The analysis of the obtained images was carried out using the public domain package ImageJ (v. 1.51k) and included automatic search for objects and their measurement by 7 indicators: area, perimeter, maximum and minimum Feret diameters, aspect ratio, circularity, and surface roundness (1-Solidity). Statistical analysis included the calculation of the minimum, maximum and mean values with a nonparametric 95% CI (bootstrap, percentile method), comparison of cultivars using the Kruskal–Wallis test, and search for the most typical objects based on the results of a between-group PCA.Results. It was shown how the size and shape indicators from the ImageJ package related to classical measurements in pomology, including length, diameter, and berry shape index. For all indicators, the differences between cultivars were highly statistically significant (p < 0.001). The prospects of using the surface roughness index for quantitative characterization of the degree of deviation of fruits from their natural shape due to mechanical and other deformations are discussed.Conclusion. The results of the automatic image analysis in the ImageJ package can be used in horticulture, breeding, and production of fruits and berries

Cytokine profile in community-acquired pneumonia in children

Community-acquired pneumonia (CAP) is one of the most common acute infectious diseases. To date, the incidence of CAP among children was decreased significantly worldwide, mainly due to increasing use of effective preventive measures. Nevertheless, CAP remains a common cause of childhood morbidity and mortality. Pneumonia may develop at any age, but most often it occurs in young children, who are more likely to have a more severe course of pneumonia. Currently, early diagnosis and prognosis of the disease severity in children is an urgent issue. It was found that, in most cases, a panel of conventional biomarkers, including the number of leukocytes, procalcitonin, CRP is not sufficient for the diagnosis of pediatric CAP. There is a demand for new biological markers which, along with clinical evaluation, may significantly improve diagnostics and management of CAP in children, thus reducing the risk of adverse outcomes associated with this disease. Such markers could be found among the cytokines, which are active participants in the CAP pathogenesis. The aim of this study was to determine the level of several cytokines in blood serum of children with CAP and to assess changes in the cytokine profile depending on the patient’s age and severity of the disease. The study included 117 children aged 1 to 18 years with a diagnosis of CAP confirmed by X-ray examination. The comparison group included 28 healthy children who did not have CAP or other signs of acute respiratory viral infection at the time of examination, being free of any chronic pathology requiring outpatient observation. A number of cytokines were determined quantitatively in blood serum, i.e., IL-1β, IFNγ, IL-2, IL-4, IL-6, IL-10, TNFα, IFNλ2 (IL-28A), IFNλ3 (IL-28B), IL-8, MCP-1, IL-17AF, GM-CSF using test systems based on the “sandwich” method of solid-phase ELISA using peroxidase labeling. As a result, it was found that the content of IL-6, IL-17AF, IL-1β, IFNγ, MCP-1, IFNλ2 (IL-28A), IFNλ3 (IL-28B), GM-CSF was significantly higher in the group of children with severe community-acquired pneumonia. The levels of certain cytokines, e.g., IL-6, IFNλ2 (IL-28A), IFNλ3 (IL-28B), GM-CSF varied depending on the age of patients, thus, probably, reflecting the degree of immune system activation in the children of different age groups

Оценка влияния полиморфизмов генов-транспортеров (RFC1, MDR1) и GGH на эффективность метотрексата при ревматоидном артрите

The efficacy of methotrexate (MT) in patients with rheumatoid arthritis (RA) may be determined by genetic factors.Objective: to evaluate the isolated and combined effects of single nucleotide polymorphisms (SNPs) of membrane transporter proteins (RFC1 80G>A and MDR1 3435C>T) and the GGH -401C>T gamma-glutamyl hydrolase enzyme genes on the efficacy of MT in patients with RA.Material and methods. The study group consisted of 85 patients with a confirmed diagnosis of RA, who received therapy with MT starting at 10 mg/week and increasing in dose to a maximum of 25 mg/week. Efficacy was assessed after six months of treatment using the dynamics of the DAS28 index, identifying patients who responded and those who did not respond to MT therapy.Genotyping of RFC1, MDR1 and GGH gene polymorphisms was performed by real-time polymerase chain reaction. Three different approaches were used to analyze the results: 1) analysis for each of the genes; 2) logistic regression; and 3) multifactor dimensionality reduction (MDR).Results and discussion. Single gene analysis was used to determine the most likely predictors of non-response to therapy: 1) for GGH-401C>T, TT genotype (odds ratio, OR 5.09; 95% confidence interval, C11.11—23.3); 2) forMDR13435C>T, the TT genotype (OR 2.38; 95% CI0.89-6.37); 3) for RFC180G>A, not - AA genotype (OR 1.87; 95% CI 0.93-3.76).The logistic regression model showed a significant effect of homozygous genotype GGH -401TT on the efficacy of MT with low sensitivity of the method. The multifactorial dimensionality reduction results show a significant synergistic effect of the MT transport genes (MDR1, RFC1) and the GGH enzyme encoding the conversion of MT to the elimination form.Conclusion. Using various statistical methods, the following results were obtained: Single gene analysis revealed the most likely predictors of nonresponse to MT therapy: GGH -401C>T - TT genotype, MDR1 3435C>T - TT genotype, RFC1 80G>A - not-AA genotype; the method of multiple logistic regression allowed to determine the significant effect of GGH -401ТТ genotype on the effect of the drug with a low sensitivity of the method; the isolated effect of polymorphisms is probably less pronounced than their combined effect on the effectiveness of MT. SNP synergism is a major contributor to the development of treatment resistance. MDR is a promising method that can be used in the future to assess the impact of SNPs.Эффективность метотрексата (МТ) у больных ревматоидным артритом (РА) может определяться генетическими факторами.Цель исследования — оценить изолированное и совместное влияние однонуклеотидных полиморфизмов (SNP) генов мембранных белков-транспортеров (RFC1 80G>A и MDR1 3435C>T) и гена фермента гамма-глутамилгидролазы GGH -401C>T на эффективность МТ у больных РА.Материал и методы. Исследуемую группу составили 85 больных с достоверным диагнозом РА, которым проводилась терапия МТ начиная с 10 мг/нед с повышением дозы максимально до 25 мг/нед. Эффективность оценивалась после 6 мес лечения по динамике индекса DAS28, были определены пациенты, отвечающие и не отвечающие на терапию МТ.Генотипирование полиморфизмов генов RFC1, MDR1 и GGH выполнено методом полимеразной цепной реакции в режиме реального времени. Для анализа результатов использовались три различных подхода: 1) анализ по каждому из генов; 2) логистическая регрессия и 3) многофакторное снижение размерности (Multifactor Dimensionality Reduction, MDR).Результаты и обсуждение. С помощью анализа по отдельным генам идентифицированы наиболее вероятные предикторы отсутствия ответа на терапию: 1) для GGH-401C>T — генотип TT(отношение шансов, ОШ5,09; 95% Доверительный интервал, ДИ 1,11—23,3); 2) для MDR13435C>T-генотип TT(ОШ2,38; 95% ДИ0,89-6,37); 3) Для RFC180G>A - генотип не АА (ОШ 1,87; 95% ДИ 0,93-3,76).Модель логистической регрессии свидетельствует о значимом влиянии на эффективность МТ носительства гомозиготного генотипа GGH -401TT при низкой чувствительности метода. Результаты многофакторного снижения размерности демонстрируют значимое синергичное влияние генов транспорта МТ (MDR1, RFC1) и фермента GGH, кодирующего конверсию МТ в форму Для элиминации.Заключение. С помощью разных статистических методов получены следующие результаты: анализ по отдельным генам выявил наиболее вероятные предикторы отсутствия ответа на терапию МТ: GGH - 401C>T - генотип TT, MDR1 3435C>T - генотип TT, RFC1 80G>A - генотип не AA; метод множественной логистической регрессии позволил определить значимое влияние генотипа GGH - 401ТТ на эффект препарата при низкой чувствительности метода; изолированное влияние полиморфизмов, вероятно, менее выражено, чем их совместное воздействие на эффективность МТ. Синергизм SNP вносит большой вклад в формирование резистентности к терапии. MDR - перспективный метод, который может быть использован в дальнейшем для оценки влияния SNP

SOCIETY OF ENVIRONMENTAL NICHE: ORAL CAVITY OF THE HEALTHY CHILDREN

In recent years, saliva is increasingly being used as a diagnostic fluid for the evaluation of various biological parameters, namely, the levels of activity of the information signal molecules of the metaorganism — the immune-neuroendocrine nature, but less often the metabolites of the microbial community and the structure of the bacterial society. The paper assesses the microbial society of the oral cavity (saliva/smear from the surfaces of the microbiota) healthy children in order to create microbial images of «health» — control that can be used in the study of the microbial community in the development of local and/or systemic pathophysiological processes, including infections, in the child’s body. Using the method of Gas chromatography mass spectrometry of microbial markers, specific chemical markers of 38 taxa of microorganisms in the oral cavity of healthy children from 1.5 to 14 years have been determined. To determine the distribution of various representatives of microbial societies between ecological niches (saliva/smear) in the oral cavity and assess the effect on them of the age of children, a Canonical Correspondences Analysis was used. A high similarity of the microbiota structure of saliva and smear from microbiota living surfaces in healthy children was found, which may indicate cross paths of bacterial representatives of different species and genera of the microbial community, or their functional plasticity. Of greatest interest are the data on the number of bacteria of the genus Alcaligenes spp. in the smear from the surfaces of the microbiota, which is twice higher, than in saliva. Alcaligenes presents itself as a professional organizer of security measures in relation to the place of residence: it produces antibiotics and original antibacterial components that disorganize the growth of a wide variety of bacteria. In addition, it is able to initiate B-lymphocytes of lymphoid follicles to produce Alcaligenes-specific antibodies, to create from them their own «cloaking» coating, facilitating its entry into Peyer’s plaques through M-cells. It can be assumed that the level of Alcaligenes spp. in saliva to some extent reflects the migration of representatives of this genus, both from the palatine and from the nasopharyngeal tonsils. The age features of the microbiota of the ecological niche — the oral cavity are determined: the number of representatives of the genus Clostridium spp. increases with age in children. And the number of bifidobacteria decreases. The results obtained by us can be used as a control in systemic pathophysiological processes, including infectious etiology, as well as during therapy

AGE AND GENDER PECULIARITIES OF BLOOD LIPIDS IN URBAN RESIDENTS (CROSS-SECTIONAL STUDY RESULTS)

Aim. To analyze the results of measurements of the blood lipid spectrum of urban  residents of the European part of the Russian Federation.Material and methods. The results of single assessment of lipid profiles in 22,436 males and 35,100 females at the age from 13 to 94 years in 347 cities and towns  of the European part of the Russian Federation were analyzed. Lipid assays were performed by the Laboratory Service «HELIX» from 28 Jan 2015 to 19 Mar 2016.Statistical methods included descriptive statistics, the analysis of distributions of sample comparisons and the search of dependencies.Results. Results of 56,235 measurements of total cholesterol (TC), 22,641 – triglycerides (TG), 21,032 – high density lipoproteins cholesterol (HDL-C) and 22,441 – low density lipoprotein cholesterol (LDL-C) were analyzed. TC above 5.2 mmol/l was identified in 55.78% samples; the proportion of people with LDL-C level above 3.0 mmol/l was 62.95%. The level of TC was the highest at the age of 43 to 62 years in both men and women, while the peak in the increase in TC in men was approximately 10 years earlier, than  in women. In patients of older age groups, TC levels decreased and reached minimum values in men and women of senile age and long-livers. Low level of HDL-C was observed in 24.46% of the total number of samples in men and in 17.68% – in women. The monotonous increase in the average levels of HDL-C was revealed in men with aging. In women the HDL-C levels increased sharply from a minimum at the age of 13 years to a maximum at the age of 25 years with a slight monotonous decrease in the following age periods. However, even in the age older than 83 years levels of HDL-C remained high (1.49 mmol/l in average). The level of TG showed significant inter-age fluctuations, especially in men. The levels of TG above 1.7 mmol/l were recorded in 30.07% of all cases.Conclusion. Two-factor analysis of variance for all lipid parameters revealed a high statistical significance of gender and age